ModG

=Introduction to ModG= ModG is a cytoplasmic molybdate-binding protein exclusive to the aerobic nitrogen-fixer Azobacter vinelandii. Molybdate is a molybdenum oxyanion (MoO42-). The group 6 element molybdenum is required by many enzymes that catalyze reactions associated with carbon, nitrogen, or sulfur metabolism. It is also part of the cofactor of the molybdoenzyme ModG. Not surprisingly, studies have linked ModG to molybdenum homeostasis within the cell.

=Structure=  The molybdoenzyme is a homotrimer. It can bind up to 8 molybdate molecules between 4 different types of active sites on subunit interfaces (BS1, BS1’, BS2, and BS2’). Binding site 1 and binding site 2 are found at opposite ends of the protein; binding site 1’ and binding site 2’ are found off-axis near like ends. The sites are connected by hydrogen bonds and thus a cooperative binding mechanism has been proposed for ModG whereby ligand engagement with type 2 sites induces conformational changes to asparagine residues at type 1 sites, reading the site for ligand interactions. The structure of ModG was solved by Delarbe et al. using multi-wavelength anomalous dispersion (MAD). Crystallization required salt-free conditions established with polyethylene glycol (PEG), at which point the authors solved the PEG crystal form using molecular replacement.

The protein is made up of 3 identical subunits that have 67 amino acid pairs and are 14.3 kDa in size. In trimer form the subunits are perpendicular to each other and intersect at the 3-fold axis (as seen in the crystallized form). Each subunit is composed of two β-barrel domains (Domain I and Domain II) that each feature a short 310-helix. Each β-barrel domain includes five antiparallel β-strands arranged in a Greek Key motif that is capped by two-turn α-helices. The folding arrangement of the ModG domains is that of an oligomer-binding (OB) fold, characteristic of toxins and other intracellular oxyanion-binding proteins. In trimerization, N and C termini (both found in Domain I) of a subunit interact with the β5-β6 loop (Domain II) of the adjoining subunit by sharing antiparallel β-sheets. Approximately 40% of monomer surface area is buried when the protein is in trimer form.

=Properties and binding affinity= About 60% of each subunit interface is non-polar (40% is polar); furthermore, there is an unequal charge distribution that is attributed to the presence of 4 side chains. These include: Lys60, Lys132, Arg6, and Arg78. The lysine residues are both found on the type 2 binding sites and are entirely buried in the protein; the arginine residues are partly exposed. This leaves the interface particularly electropositive; electrostatic repulsion is balanced by several favorable interactions that include the formation of 24 hydrogen bonds, 2 salt bridges and approximately 24% of total hydrophobic surface buried (per subunit). The trimer is further stabilized by the binding of an oxyanion which would have a neutralizing effect.

Type 1 binding sites utilize hydrogen bonds and interactions with uncharged polar residues to bind molybdate. The tight pocket volume and rigidity of the site suggest that there is high selectivity for molybdate (or tungstate because ModG cannot differentiate between the two oxyanions). Type 2 sites are comparatively larger in pocket volume, supple, and feature electropositive lysine residues that would contribute to high affinity for negatively charged oxyanions like molybdate. Because the type 2 sites are larger and more flexible, there would be less specificity for molybdate and thus other oxyanions such as phosphate would likely compete for binding.

=Function in Bacteria= The ability of A. vinelandii to differentiate between molybdate and other oxyanions is of particular interest to researchers. Phosphate is distinguished from molybdate by the divergence in protonation states: phosphate is protonated at physiological pH whereas molybdate is not. The distinction between sulfate and molybdate is reliant upon differences in ligand size. Interestingly, molybdate permeases are not able to distinguish between tungstate and molybdate. Regulation of these intracellular metabolites is attributed to other molybdate-binding proteins collectively known as molbindins.

=Fate= ModG is eventually degraded and incorporated into molybdopterin, a cofactor of molybdenum enzymes, or the iron-molybdenum cofactor of a nitrogenase enzyme. Currently there is little known about cofactor biosynthesis involving the ModG protein.

=References=

=See also=

This page originally authored by Corbin Black